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Cell Cycle Regulation and Apoptotic Responses of the Embryonic Chick Retina by Ionizing Radiation.

Mayer M, Kaiser N, Layer PG, Frohns F - PLoS ONE (2016)

Bottom Line: Our studies reveal a lack in the radiation-induced activation of a G1/S checkpoint, but rapid abrogation of G2/M progression after IR in retinal progenitors throughout development.Whereas the general sensitivity towards RIA declined with ongoing differentiation, its dose dependency constantly increased with age.For all embryonic stages RIA occurred during comparable periods after irradiation, but in older animals its maximum shifted towards earlier post-irradiation time points.

View Article: PubMed Central - PubMed

Affiliation: Developmental Biology and Neurogenetics, Darmstadt University of Technology, Darmstadt, Germany.

ABSTRACT
Ionizing radiation (IR) exerts deleterious effects on the developing brain, since proliferative neuronal progenitor cells are highly sensitive to IR-induced DNA damage. Assuming a radiation response that is comparable to mammals, the chick embryo would represent a lower vertebrate model system that allows analysis of the mechanisms underlying this sensitivity, thereby contributing to the reduction, refinement and replacement of animal experiments. Thus, this study aimed to elucidate the radiation response of the embryonic chick retina in three selected embryonic stages. Our studies reveal a lack in the radiation-induced activation of a G1/S checkpoint, but rapid abrogation of G2/M progression after IR in retinal progenitors throughout development. Unlike cell cycle control, radiation-induced apoptosis (RIA) showed strong variations between its extent, dose dependency and temporal occurrence. Whereas the general sensitivity towards RIA declined with ongoing differentiation, its dose dependency constantly increased with age. For all embryonic stages RIA occurred during comparable periods after irradiation, but in older animals its maximum shifted towards earlier post-irradiation time points. In summary, our results are in good agreement with data from the developing rodent retina, strengthening the suitability of the chick embryo for the analysis of the radiation response in the developing central nervous system.

No MeSH data available.


Related in: MedlinePlus

X-ray-induced cell cycle arrest at G2/M-checkpoint in embryonic chick retina is abrogated at about 3 hours post-irradiation throughout development.(A-F) Staining against the mitotic marker pH3 (red) in control (A-C) and 2 Gy (D-F) irradiated E3 retinae at 1, 3 and 6 hrs after irradiation. Nuclei were counterstained with DAPI (blue). Note absence of mitotic cells up to 3 hrs after irradiation. (G) Mitotic index in retinae of E3 embryos after irradiation with various doses at different time points. (H) Absolute numbers of mitotic events in retinal slices of E5 after irradiation with various doses at different time points. (I) Absolute numbers of mitotic events in retinal slices of E7 after irradiation with various doses at different time points. Note that at all stages, cells are released from G2-arrest around 3 hrs after irradiation. Data are presented as means (n = 3, with at least 100 cell analyzed for E3 and at least three different retinal slices analyzed for E5 and E7) ± SEM. (*P<0.05 **P< 0.01 *** P<0.001). Scale bar = 25 μm. RPE, retinal pigmented epithelium; pONL, presumptive outer nuclear layer.
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pone.0155093.g003: X-ray-induced cell cycle arrest at G2/M-checkpoint in embryonic chick retina is abrogated at about 3 hours post-irradiation throughout development.(A-F) Staining against the mitotic marker pH3 (red) in control (A-C) and 2 Gy (D-F) irradiated E3 retinae at 1, 3 and 6 hrs after irradiation. Nuclei were counterstained with DAPI (blue). Note absence of mitotic cells up to 3 hrs after irradiation. (G) Mitotic index in retinae of E3 embryos after irradiation with various doses at different time points. (H) Absolute numbers of mitotic events in retinal slices of E5 after irradiation with various doses at different time points. (I) Absolute numbers of mitotic events in retinal slices of E7 after irradiation with various doses at different time points. Note that at all stages, cells are released from G2-arrest around 3 hrs after irradiation. Data are presented as means (n = 3, with at least 100 cell analyzed for E3 and at least three different retinal slices analyzed for E5 and E7) ± SEM. (*P<0.05 **P< 0.01 *** P<0.001). Scale bar = 25 μm. RPE, retinal pigmented epithelium; pONL, presumptive outer nuclear layer.

Mentions: To rule out earlier changes in S-phase entry, E7 embryos were double-labeled with EdU and BrdU. Hereby, EdU was administered directly after irradiation and BrdU was added in excess at 3 hrs. Fixation was done at 6 hrs after irradition (Fig 3D). No significant changes between both EdU (p-value = 0.739) and BrdU incorporation (p-value = 0.68) was detected between controls (17% for EdU, 13% for BrdU) and 2 Gy irradiated samples (16% for EdU and 10% for BrdU) (Fig 2D and 2E). FACS analysis at 6 hrs after the irradiation also showed no radiation-induced changes in cell cycle distribution (Fig 2F, see S2B Fig for FACS blots).


Cell Cycle Regulation and Apoptotic Responses of the Embryonic Chick Retina by Ionizing Radiation.

Mayer M, Kaiser N, Layer PG, Frohns F - PLoS ONE (2016)

X-ray-induced cell cycle arrest at G2/M-checkpoint in embryonic chick retina is abrogated at about 3 hours post-irradiation throughout development.(A-F) Staining against the mitotic marker pH3 (red) in control (A-C) and 2 Gy (D-F) irradiated E3 retinae at 1, 3 and 6 hrs after irradiation. Nuclei were counterstained with DAPI (blue). Note absence of mitotic cells up to 3 hrs after irradiation. (G) Mitotic index in retinae of E3 embryos after irradiation with various doses at different time points. (H) Absolute numbers of mitotic events in retinal slices of E5 after irradiation with various doses at different time points. (I) Absolute numbers of mitotic events in retinal slices of E7 after irradiation with various doses at different time points. Note that at all stages, cells are released from G2-arrest around 3 hrs after irradiation. Data are presented as means (n = 3, with at least 100 cell analyzed for E3 and at least three different retinal slices analyzed for E5 and E7) ± SEM. (*P<0.05 **P< 0.01 *** P<0.001). Scale bar = 25 μm. RPE, retinal pigmented epithelium; pONL, presumptive outer nuclear layer.
© Copyright Policy
Related In: Results  -  Collection

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pone.0155093.g003: X-ray-induced cell cycle arrest at G2/M-checkpoint in embryonic chick retina is abrogated at about 3 hours post-irradiation throughout development.(A-F) Staining against the mitotic marker pH3 (red) in control (A-C) and 2 Gy (D-F) irradiated E3 retinae at 1, 3 and 6 hrs after irradiation. Nuclei were counterstained with DAPI (blue). Note absence of mitotic cells up to 3 hrs after irradiation. (G) Mitotic index in retinae of E3 embryos after irradiation with various doses at different time points. (H) Absolute numbers of mitotic events in retinal slices of E5 after irradiation with various doses at different time points. (I) Absolute numbers of mitotic events in retinal slices of E7 after irradiation with various doses at different time points. Note that at all stages, cells are released from G2-arrest around 3 hrs after irradiation. Data are presented as means (n = 3, with at least 100 cell analyzed for E3 and at least three different retinal slices analyzed for E5 and E7) ± SEM. (*P<0.05 **P< 0.01 *** P<0.001). Scale bar = 25 μm. RPE, retinal pigmented epithelium; pONL, presumptive outer nuclear layer.
Mentions: To rule out earlier changes in S-phase entry, E7 embryos were double-labeled with EdU and BrdU. Hereby, EdU was administered directly after irradiation and BrdU was added in excess at 3 hrs. Fixation was done at 6 hrs after irradition (Fig 3D). No significant changes between both EdU (p-value = 0.739) and BrdU incorporation (p-value = 0.68) was detected between controls (17% for EdU, 13% for BrdU) and 2 Gy irradiated samples (16% for EdU and 10% for BrdU) (Fig 2D and 2E). FACS analysis at 6 hrs after the irradiation also showed no radiation-induced changes in cell cycle distribution (Fig 2F, see S2B Fig for FACS blots).

Bottom Line: Our studies reveal a lack in the radiation-induced activation of a G1/S checkpoint, but rapid abrogation of G2/M progression after IR in retinal progenitors throughout development.Whereas the general sensitivity towards RIA declined with ongoing differentiation, its dose dependency constantly increased with age.For all embryonic stages RIA occurred during comparable periods after irradiation, but in older animals its maximum shifted towards earlier post-irradiation time points.

View Article: PubMed Central - PubMed

Affiliation: Developmental Biology and Neurogenetics, Darmstadt University of Technology, Darmstadt, Germany.

ABSTRACT
Ionizing radiation (IR) exerts deleterious effects on the developing brain, since proliferative neuronal progenitor cells are highly sensitive to IR-induced DNA damage. Assuming a radiation response that is comparable to mammals, the chick embryo would represent a lower vertebrate model system that allows analysis of the mechanisms underlying this sensitivity, thereby contributing to the reduction, refinement and replacement of animal experiments. Thus, this study aimed to elucidate the radiation response of the embryonic chick retina in three selected embryonic stages. Our studies reveal a lack in the radiation-induced activation of a G1/S checkpoint, but rapid abrogation of G2/M progression after IR in retinal progenitors throughout development. Unlike cell cycle control, radiation-induced apoptosis (RIA) showed strong variations between its extent, dose dependency and temporal occurrence. Whereas the general sensitivity towards RIA declined with ongoing differentiation, its dose dependency constantly increased with age. For all embryonic stages RIA occurred during comparable periods after irradiation, but in older animals its maximum shifted towards earlier post-irradiation time points. In summary, our results are in good agreement with data from the developing rodent retina, strengthening the suitability of the chick embryo for the analysis of the radiation response in the developing central nervous system.

No MeSH data available.


Related in: MedlinePlus